Ubiquitin-targeted bacterial effectors: rule breakers of the ubiquitin system

Abstract

Regulation through post-translational ubiquitin signaling underlies a large portion of eukaryotic biology. This has not gone unnoticed by invading pathogens, many of which have evolved mechanisms to manipulate or subvert the host ubiquitin system. Bacteria are particularly adept at this and rely heavily upon ubiquitin-targeted virulence factors for invasion and replication. Despite lacking a conventional ubiquitin system of their own, many bacterial ubiquitin regulators loosely follow the structural and mechanistic rules established by eukaryotic ubiquitin machinery. Others completely break these rules and have evolved novel structural folds, exhibit distinct mechanisms of regulation, or catalyze foreign ubiquitin modifications. Studying these interactions can not only reveal important aspects of bacterial pathogenesis but also shed light on unexplored areas of ubiquitin signaling and regulation. In this review, we discuss the methods by which bacteria manipulate host ubiquitin and highlight aspects that follow or break the rules of ubiquitination.

Keywords: Ubiquitin; bacterial effector; bacterial pathogenesis; post-translational modification.

Figure 1. Cysteine‐independent ubiquitin ligases

(A) Structure of the human U‐box E3 ligase UBE4B (PDB 3L1Z; Benirschke et al, 2010), with regions involved in E2 binding and E2 activation colored in blue and gold, respectively. (B) Structure of the Pseudomonas syringae U‐box E3 ligase AvrProB (PDB 2FD4; Janjusevic et al, 2006), highlighting conserved regions for E2 binding and activation. (C) Structure of the Xanthomonas campestris XL‐box E3 ligase (PDB 4FC9; Singer et al, 2013), highlighting its distinctive fold and E2 binding. (D) Mechanism of RING/U‐box‐type E3 activation through directing a dynamic E2~Ub conjugate toward a closed and reactive conformation (Pruneda et al, , ; Dou et al, ; Plechanovová et al, 2012). Bacterial followers of this mechanism include U‐box effectors. (E) Mechanism of multicomponent SCF E3 ligase complexes, highlighting the versatility imparted by F‐box substrate adaptors (Baek et al, 2020). Bacterial followers of this mechanism include F‐box effectors. (F) Mechanism of SidE noncanonical ubiquitination, whereby an mART domain consumes NAD to ADP‐ribosylate Arg42 of ubiquitin (releasing NAM), which is then activated onto a PDE domain (releasing AMP) and subsequently transferred onto a substrate serine residue (Bhogaraju et al, ; Qiu et al, 2016).

Figure 2. Cysteine‐dependent ubiquitin ligases

(A) Structure of the human HECT E3 ligase NEDD4L (PDB 3JVZ; Kamadurai et al, 2009), with the E2‐binding region and active site colored in blue and gold, respectively. (B) Structure of the Salmonella Typhimurium HECT‐like E3 ligase SopA (PDB 2QYU; Diao et al, 2008), highlighting topologically analogous E2‐binding region and active site. (C) Structure of the Salmonella Typhimurium NEL SspH2 (PDB 3G06; Quezada et al, 2009), highlighting its distinctive tri‐lobed fold (N‐, M‐, and C‐lobe), E2‐binding region, and active site. (D) Mechanism of HECT E3 ligases, highlighting flexibly‐linked N‐ and C‐lobes that facilitate docking and transfer of ubiquitin from an E2, as well as activation of ubiquitin for substrate ubiquitination and/or polyUb chain formation (Kamadurai et al, ; Jäckl et al, ; preprint: Franklin et al, 2023). (E) Mechanism of bacterial HECT‐like E3 ligases, highlighting common themes including interlobe flexibility and E2 transthiolation (Diao et al, ; Lin et al, 2012). (F) Mechanism of bacterial SNL E3 ligases, highlighting distinctive bilobed topology and E2~Ub binding (Hsu et al, ; Wasilko et al, 2018).

Figure 3. CA‐clan deubiquitinases

(A) Structure of the human OTU deubiquitinase OTULIN (PDB 3ZNV; Keusekotten et al, ; Rivkin et al, 2013), with Ub‐binding sites and the catalytic triad colored in blue and gold, respectively. (B) Structure of the Legionella pneumophila OTU deubiquitinase LotC (PDB 7BU0; Liu et al, ; Shin et al, 2020a), with analogous Ub‐binding sites and catalytic triad colored in blue and gold, respectively. (C) Structure of the Legionella pneumophila deubiquitinase RavD (PDB 6NII; Wan et al, 2019b), highlighting its distinctive CA‐clan fold yet related Ub‐binding regions (blue) and catalytic triad (gold). (D) Mechanism of diUb proteolysis by eukaryotic OTU deubiquitinases (Mevissen & Komander, 2017). (E) Mechanism of diUb proteolysis by bacterial OTU deubiquitinases, highlighting a common route with distinctive features (Schubert et al, 2020). (F) Mechanism of UBE2N inactivation by Legionella pneumophila MavC, a CA‐clan effector that has evolved transglutaminase function. MavC forms a thioester intermediate with the ubiquitin Gln40 side chain before facilitating transfer onto a UBE2N lysine residue (Gan et al, 2019a). This process is reversed by the related Legionella pneumophila effector, MvcA (Gan et al, 2020).

Figure 4. CE‐clan Ub/Ubl proteases

(A) Structure of the human NEDD8 protease NEDP1 (PDB 2BKR; Reverter et al, ; Shen et al, 2005), with S1 site variable regions (VR1‐3) and catalytic triad colored in blue and gold, respectively. (B) Structure of the Legionella pneumophila CE‐clan deubiquitinase SdeA (PDB 5CRB; Sheedlo et al, 2015), highlighting an analogous S1 site and catalytic triad in blue and gold, respectively. (C) Structure of the Chlamydia trachomatis CE‐clan deubiquitinase/acetyltransferase ChlaDUB1 (PDB 6GZT; Pruneda et al, 2018), highlighting an analogous Ub‐binding S1 site (blue) but distinctive coenzyme A‐binding site (teal) that endows moonlighting functionalities. (D) Mechanism of NEDD8 proteolysis by Ub‐like proteases (Reverter et al, ; Shen et al, 2005). (E) Mechanism of diUb proteolysis by bacterial CE‐clan deubiquitinases, highlighting a common route with distinctive substrate‐binding sites (Sheedlo et al, ; Pruneda et al, 2016). (F) Mechanism of acetylation by bacterial CE‐clan acetyltransferases, typified by Chlamydia trachomatis ChlaDUB1 (Pruneda et al, 2018). Acetylation proceeds via a “ping‐pong” mechanism wherein an acetyl group is transferred onto the active site cysteine before modifying a target serine, threonine, or lysine residue (Mukherjee et al, 2006).

Figure 5. Regulation of and by ubiquitin

(A) Structure of the insect Ub kinase PINK1 (green) bound to Ub (red; PDB 6EQI; Schubert et al, 2017), highlighting proximity of Ub Ser65 to the PINK1 active site. (B) Structure of the Legionella pneumophila SdeA mART domain (PDB 5YIJ; Akturk et al, ; Dong et al, ; Kalayil et al, ; Kim et al, ; Wang et al, 2018), highlighting proximity of Ub Arg42 to the mART active site. (C) Structure of the Burkholderia pseudomallei Ub/NEDD8 deamidase CHBP (PDB 4HCN; Yao et al, 2012), highlighting proximity of Ub Gln40 to the CHBP active site. (D) Mechanism of targeted protein degradation via the ubiquitin–proteasome system. (E) Mechanism of hijacking the host ubiquitin–proteasome system by bacterial metaeffector E3 ligases (Kubori et al, 2010). (F) Mechanism of dual ubiquitination/phosphorylation regulation by the Shigella flexneri effector kinase OspG, wherein OspG stabilizes E2 ~ Ub conjugates in an inactive conformation while simultaneously stabilizing an active OspG conformation for phosphorylation (Pruneda et al, 2014).